An initial event in the keratinization of squamous epithelial cells is the deposition of a protein layer 15 nm thick along the cytoplasmic face of the plasma membrane to form the cell envelope (CE). The stability of this highly resistant structure which, is essential for the protective function of the cornfield layer, is primarily due to the formation of epsilon-(gamma-glutamyl-lysine) bonds. Considerable data from numerous investigators suggest that CE have a high cystine content. Furthermore, dense homogeneous deposits, which have a sulfur-rich component, are thought to participate in the formation of CE. However, to date, conclusive data identifying this cystine-rich component has not been reported. Evidence is presented in this proposal for the isolation and characterization of a cDNA clone encoding a previously undescribed protein which is a major component of the CE. The high cystine content of this protein (6.9%) and its apparent localization with granules suggest that it may be the sulfur-rich component of dense homogeneous deposits. Transcripts of the gene encoding this protein, referred to as major cell envelope protein (MCEP), are restricted to the granular layer of the epidermis and are as abundant as those of the differentiation-specific keratins, K1 and K10, and filaggrin. Expression of the MCEP gene occurs, not only in the epidermis, but in all keratinizing squamous epithelia. In addition, immunological studies with antibodies elicited against a specific mouse MCEP peptide recognize conserved epitopes in man and all tested vertebrates. Thus, MCEP is highly conserved and, quantitatively, the most important CE component discovered to date. The major role played by this protein in CE formation and function makes the gene encoding it a likely target for mutation resulting in keratinization disorders. The genes encoding mouse and human MCEP will be isolated and sequenced. Due to deficiencies in existing cell culture systems, experiments designed to identify sequences responsible for tissue-, developmental-, and differentiation-specific expression of the MCEP gene must be performed in transgenic mice. After determining sequences important for appropriate expression, proteins interacting with these sequences will be characterized and the genes encoding these proteins will be cloned. Mutations designed to interface with the assembly of MCEP into CE, its function and expression will be introduced into the germline of mice to determine if defects in keratinization results. CE formed in transgenic mice containing mutant MCEP genes will be examined by ultrastructural electron microscopic methods to determine the process of assembly. Finally, existing mouse and human mutations exhibiting keratinization disorders will be examined to determine if there is direct involvement of the MCEP gene.